PCR (Polymerase Chain Reaction) is a commonly used method to make multiple copies of a DNA (Deoxyribonucleic Acid) sequence for various applications such as DNA cloning for sequencing, diagnosing disease, identifying individuals from DNA samples, and performing functional analyses of genes. In PCR, replication of the DNA sequence takes place in multiple thermal cycles, with each cycle typically having three main steps: denaturation, annealing and extension. In the denaturation step, a double-stranded DNA template is heated to about 94-98° C. for 20-30 seconds to yield single-stranded DNA. In the annealing step, primers are annealed to the single-stranded DNA by lowering the temperature to about 50-65° C. for 20-40 seconds. In the extension step, using a DNA polymerase (such as Taq), a new double-stranded DNA is synthesized by extending the primer that has been annealed to the single-stranded DNA at an optimum activity temperature of the DNA polymerase (75-80° C. for Taq). In addition to the three main steps mentioned above, an initialization step may be required if the DNA polymerase used is heat activated, and the final extension step of the last cycle may be held for a longer period of time (e.g. 5-15 minutes) to ensure that there are no remaining single-stranded DNA fragments.
Any device for performing PCR needs to be able to perform the repeated thermal cycles in order for the steps of denaturation, annealing and extension to take place. This involves heating and cooling the reaction to the required temperatures and holding the required temperatures for the necessary lengths of time. Given that temperatures go up to nearly and/or more than 100° C., existing microfluidic or lab-on-chip PCR devices typically require an external thermal cycler to supply the necessary heat, thereby limiting their true portability and size during use.
DNA replication via PCR is exponential as the new double-stranded DNA formed in a cycle undergoes denaturation, annealing and extension in the next cycle, such that each cycle effectively doubles the number of DNA sequences obtained. The number of PCR amplification cycles is thus dependent on the number of copies of the DNA at the start of the reaction.
By contrast, digital PCR is a form of PCR where the amount of DNA in the initial sample can be determined without being dependent on the number of PCR cycles so that the starting amount of DNA can be quantified without having to rely on uncertain exponential data. In digital PCR, one DNA sample is partitioned into multiple wells and the PCR is performed simultaneously in all the wells. Concentration of DNA within the multiple wells is assumed to follow a Poisson Distribution. After PCR has been performed, each well will indicate either absence (negative) or presence (positive) of amplified DNA therein. In this way, absolute quantification may be achieved by counting the number of wells that show a positive end result.
Currently, digital PCR systems, like conventional PCR systems, also rely on external thermal cyclers to supply the necessary heat to each of the wells into which the DNA is partitioned, thereby similarly limiting their size and portability. This limits the ability of health care workers in many parts of the world to efficiently perform important infectious disease diagnoses to control epidemics, particularly where the population is spread out over large areas and access to the testing equipment is hampered by poor infrastructure and transport networks.
Recently, a dc heater has been developed that can be heated to up to or more than 100° C. using a voltage of 9 volts or less and wherein the dc heater is one inch square or smaller. The dc heater comprises a discrete heating area made of a heat conductive material disposed on a surface that is electrically non-conductive and at least one conductive trace configured to be connected to a dc voltage source and to heat the discrete heating area to a uniform temperature when connected to the dc voltage source. The at least one conductive trace is disposed in an undulating configuration on the surface at least partially around the discrete heating area. Such a small heater powered by a low voltage source opens up possibilities for many applications in terms of space and power saving, in particular for portable and even disposable use. However, use of such a dc heater for digital PCR is still a challenge due to size constraints in providing a sufficient number of partitions or wells within a small area to contain the DNA sample therein for a meaningful reaction to be carried out when heated by such a small dc heater.